| ObjectiveTo investigate the combinated inhibitory effect of PARP inhibitor (HD-199 and HD-199-M) with DDP on human breast cancer cell MDA-MB-231 in vitro, in order to provide experimental basis for the treatment to Basal-like breast cancer,and hope to propose a new strategy to Basal-like breast cancer.MethodsMDA-MB-231 cells of human breast cancer were cultured in vitro, the morphological changes were observed with in verted optical microscope.Firstly,the breast cancer cell-line MDA-MB-231 was treated with HD-199(5μg/ml,15μg/ml,45μg/ml,HD-199-M(5μg/ml,15μg/ml,45μg/ml) and DDP(5μg/ml) separately for 24,48 and 72 hours to select the appropriate dosage.Then HD-199(15μg/ml,45μg/ml) and HD-199-M (15μg/ml,45μg/ml) respectively combined with DDP (2.5μg/ml) to treat MDA-MB-231 cells for 24,48 and 72 hours.MTT assay was used to investigate their inhibitory effect on the proliferation of cancer cells.The combining effect was evaluated by the Jins Formula.After MDA-MB-231 cells were incubated with HD-199(15μg/ml),HD-199-M(15μg/ml) separately or combined with DDP(2.5μg/ml) for 24,48 and 72 hours,morphologic character of apoptosis was evaluated by Hoechst 33342/PI double staining cooperating under fluorescent microscope.Lastly,apoptosis was determined by staining cells with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) and analyzed in Flow cytometry (FCM) after 24,48 and 72 hours.Results1. Inverted microscope observation shows that,the cell number of dosing group significantly reduced,cell morphology changes,cell shrinkage,formation of cell debris and particulate matter,can be seen that the cell apoptosis or necrosis of the refractive index or weak and the cell edge is not clear under microscope. With the increase of drug concentration and treatment time,gradually reduce the number of cells,showing that more necrotic and apoptotic cell.2. The HD-199 can inhibit the proliferation of human breast cancer cells MDA-MB-231 at the concentration of 5μg/ml,15μg/ml and 45μg/ml,the 24 hours of inhibition ratio were 4.54±1.20%,8.18±1.11%,10.7±1.05%;the 48 hours of inhibition ratio were 9.31±2.08±%,10.9±1.31%,12.6±2.11%;the 72 hours of inhibition ratio were 6.45±2.24%,10.9±1.92%,13.4±2.12%.The HD-199-M can inhibit the proliferation of human breast cancer cells MDA-MB-231 at the concentration of 5μg/ml,15μg/ml and 45μg/ml,the 24 hours of inhibition ratio were 10.0±2.06%,20.3±1.24%,37.1±2.19%;the 48 hours of inhibition ratio were 23.2±2.19%.29.9±3.08%.41.4±2.26%;the 72 hours of inhibition ratio were 21.2±2.16%,34.3±2.27%,55.0±1.55%.From the above dates,the inhibition of the PARP inhibitor on MDA-MB-231 cells were shown in the dose- dependent and time-dependent manner, the breast cancer cells decreased gradually with the inereasing of drug concentration and action time(P<0.01).3. DDP at the concentration of 5μg/ml signifieantly inhibited the proliferation of MDA-MB-231 cells,the OD values of 24,48 and 72 hours were 0.287±0.012,0.379±0.010,0.434±0.011,compared to the control group OD values 0.443±0.005,0.765±0.006,1.208±0.007,with the increase of treatment time,the OD values gradually decreased.the inhibition ratio of 24,48 and 72 hours were 35.2±2.37%,50.4±1.63%,64.0±2.13%,the inhibition of DDP was better than HD-199 single-agent group and HD-199-M single-agent group.4. The q values of HD-199(at the concentration of 15μg/ml) combined with DDP(at the concentration of 2.5μg/ml) of 24,48 and 72 hours were 1.36,1.32,1.25,the q values of HD-199(at the concentration of 45μg/ml) combined with DDP(at the concentration of 2.5μg/ml) of 24,48 and 72 hours were 1.39,1.36,1.27,all the q value were larger than 1.15,suggested a synergistic effect.The q values of HD-199-M(at the concentration of 15μg/ml) combined with DDP(at the concentration of 2.5μg/ml) of 24,48 and 72 hours were 0.65,1.27,1.14,the q value of 24 hours was less than 0.85,suggested an antagonistic effect,the q value of 48 hours was larger than 1.15,suggested a synergistic effect,the q value of 72 hours was in the range of 0.85 and 1.15,suggested an additive effect.The q values of HD-199-M(at the concentration of 45μg/ml) combined with DDP(at the concentration of 2.5μg/ml) of 24,48 and 72 hours were 0.95,1.26,1.15;the q value of 24 hours was in the range of 0.85 and 1.15,suggested an additive effect,the q value of 48 hours and 72 hours were larger than 1.15,suggested a synergistic effect.The inhibition of HD-199 combined with DDP was more obvious than HD-199-M combined with DDP.5. According to the results of Hoechst 33342/PI double staining cooperating with fluorescence microscope,the control group Hoechst coloration of the nucleus shape was round,light blue,darker blue granules with the nuclear chromatin is evenly distributed;part of the treatment group cells showing cell shrinkage,nucleolus reduce or disappear,cytoplasmic concentration and chromatin condensation.6. According to the results of Annexin V-FITC/PI double staining cooperating with Flow cytometry,the 24,48 and 72 hours of HD-199 single-agent group(at the concentration of 15μg/ml) apoptosis rate were 8.6±0.99%,10.5±0.92%,13.8±1.65%,the proportion of late apoptotic cells in the total apoptotic cells were 52.3%,55.2%,52.9%;the 24,48 and 72 hours of HD-199-M single-agent group(at the concentration of 15μg/ml) apoptosis rate were 10.2±1.91%,12.7±1.37%,15.0±1.54%,the proportion of late apoptotic cells in the total apoptotic cells were 51.0%,49.6%,50.7%;the 24,48h and 72 hours apoptosis rate of HD-199(at the concentration of 15μg/ml) combined with DDP(at the concentration of 2.5μg/ml) were 16.3±1.12%,17.8±1.29%,19.8±0.89%,the proportion of late apoptotic cells in the total apoptotic cells were 53.4%,57.9%,63.1%;the 24,48 and 72 hours apoptosis of HD-199-M(at the concentration of 15μg/ml) combined with DDP(at the concentration of 2.5μg/ml) 17.3±1.79%,20.1±1.33%,23.2±1.42%,the proportion of late apoptotic cells in the total apoptotic cells were 54.3%,57.7%,57.8%.After dtug treatment,in total apoptosis rate,the late apoptosis cells accounted for a large proportion.Conclusions1. HD-199 and HD-199-M can inhibit the proliferation of human breast cancer MDA-MB-231 cells in a dose-dependent and time-dependent marnner within a certain dosage range.2. DDP has a signifieantly inhibition on the proliferation of MDA-MB-231 cells,and the inhibition was better than HD-199 single-agent group and HD-199-M single-agent group.3. HD-199 and HD-199-M in a low toxic dosage combined with DDP had certain synergistic effect in suppressing the growth of human breast cancer cell MDA-MB-231.4. The synergistic effect of HD-199 and HD-199-M combined with DDP may be increase the percentage of late apoptotic cells.And in total apoptosis rate,the early and late apoptosis cells as treatment time increased,showing time-dependent,means that the effect of induction of apoptosis enhanced when PARP inhibitor combined with DDP. |
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